Nuclear Histone 3 Post translational Modification Profiling in Whole Cells using Spectral Flow Cytometry

Data Analysis

Author

Serrano Lab

Background

In this analysis workflow we use spectral flow cytometry to simultaneously assess histone H3 post-translational modifications (H3-PTMs) at the single-cell level, in the context of cell cycle status and cell-type-specific markers. This workflow was implemented in BioProtocol Exchange by Golden et al., 2024 and contains supplementary figures and data analysis associated with the pre-print.

Information about biological material

iPSC-derived Neuronal Progenitor Cells (NPCs) were generated using the female control line SCTi003-A (Stem Cell Technologies). NPC induction was performed with the STEMdiff SMADi Neural Induction Kit, following the manufacturer’s instructions. All experiments were conducted with three biological replicates, defined in this context as NPCs induced in three independent wells. For further details on experimental design and data acquisition, please refer to Golden et al., 2024.

Information about initial data processing performed in OMIQ

Data processing Strategy Additional Information
Scaling Hyperbolic arcsine (arcsinh) Finak et al., 2010
Gating Cell QC and manual gating for comparison Golden et al., 2024
Subsetting Fully stained samples Removal of single color controls and blanks to prevent noise in downstream analysis
Subsampling Random downsampling. Equal number of cells available in the Live Cells filter Braanker et al., 2021 Note: Density-dependent downsampling strategy should be tested to assess if there is loss of small cell populations due to random cell selection Qiu, 2017
Dimension Reduction PCA all features
Dimension Reduction opt-SNE selected features
Clustering FlowSOM selected features

Data analysis and visualization

The goal of this analysis is to assess H3-PTM signatures in NPCs in the context of their cell cycle status and lineage markers (PAX6 in this case).

Note

I named the fluorescence intensity column as mfi and it is misleading. Note that this is not the median value but the fluorescence value of each feature in each cell.

Summary of Data Frame
event clusterid feature mfi
Min. : 776 Length:554130 Length:554130 Min. :-1.9451
1st Qu.: 9171 Class :character Class :character 1st Qu.: 0.4495
Median :17176 Mode :character Mode :character Median : 2.2822
Mean :19285 NA NA Mean : 2.3179
3rd Qu.:28082 NA NA 3rd Qu.: 4.1336
Max. :47312 NA NA Max. : 6.8623
Summary of Data Frame with scaled values
event clusterid feature mfi mfi_z.V1
Min. : 776 Length:554130 Length:554130 Min. :-1.9451 Min. :-2.2360456
1st Qu.: 9171 Class :character Class :character 1st Qu.: 0.4495 1st Qu.:-0.9800129
Median :17176 Mode :character Mode :character Median : 2.2822 Median :-0.0186857
Mean :19285 NA NA Mean : 2.3179 Mean : 0.0000000
3rd Qu.:28082 NA NA 3rd Qu.: 4.1336 3rd Qu.: 0.9523977
Max. :47312 NA NA Max. : 6.8623 Max. : 2.3836750

Z-score column was added to data

Show code
# Create histograms for original and normalized mfi variables
par(mfrow = c(1,2))
hist(df$mfi, main = "NPC", xlab = "Initial Fluorescence values")
hist(df$mfi_z, main = "NPC", xlab = "Z-scores")
Figure 1: Assessment of data scaling

Goal: Assess H3-PTM values in the context of cell cycle status and markers such us cleaved-Caspase3 and PAX6. This section also aims to compare FlowSOM unsupervised clustering and manual gates.

Note

In the context of this project (and most spectral data) the targets of the antibodies are referred to as features. Gates, whether manual or created through FlowSOM, are referred to as filters.

Initial visualization of all events within each cluster

This plot allows visualization of single events in each cluster. Not included in Methods paper

Adapted from Heatmap for time series

Show code
p <-ggplot(df,aes(event,feature,fill=mfi_z))+
  geom_tile(color= "white",linewidth=0.000000001) + 
  scale_fill_viridis(name="Scaled Fluorescense",
                    alpha = 1,
                    begin = 0,
                    end = 1,
                    direction = 1,
                    discrete = FALSE,
                    option = "C",
                    aesthetics = "fill")
p <-p + facet_grid(~clusterid, scales = "free", space = "free",)
p <-p + scale_y_discrete(limits = rev(levels(df$feature)))   # Reverse the order of the factor levels for y-axis
p <-p + scale_x_discrete(labels = NULL) 
p <-p + theme_minimal(base_size = 8)
p <-p + labs(title= paste("Fluorescence intentisity values of each feature by fsom cluster | NPC"), x="Event", y="Feature")
p <-p + theme(legend.position = "bottom")+
  theme(plot.title=element_text(size = 14))+
  theme(axis.text.y=element_text(size=6)) +
  theme(strip.background = element_rect(colour="white"))+
  theme(plot.title=element_text(hjust=0))+
  theme(axis.ticks=element_blank())+
  theme(axis.text=element_text(size=7))+
  theme(legend.title=element_text(size=8))+
  theme(legend.text=element_text(size=6))+
  theme(strip.text.x = element_text(angle = 90, hjust = 1)) +  # Rotate the cluster id labels
  removeGrid()#ggExtra
# you will want to expand your plot screen before this bit!
p
Figure 2: Single cell resolution of feature MFI per cluster

Heatmap visualization of concatenated files

For this plot, we used heatmap data downloaded from OMIQ, which includes three concatenated biological replicates. To compare the resolution between FSOM clusters and manual gating, data will be plotted for both sets of filters.

Head of Data Frame containing unsupervised clusters
H3K14ac phH3_S10 H3K4me3 Total.H3 Caspase3 PAX6 H3K27ac H3K9ac FxCycle
fsom_02 0.45 0.24 4.80 0.80 0.03 1.25 3.00 4.12 3.57
fsom_09 0.78 0.54 5.39 1.68 -0.38 3.25 4.67 5.35 4.20
fsom_04 0.47 0.23 4.85 0.93 1.42 2.14 3.30 4.34 3.63
fsom_01 0.53 0.24 5.10 1.23 -0.15 1.24 3.74 4.52 3.78
fsom_05 0.79 0.37 5.44 1.69 -0.35 1.78 4.44 5.24 4.23
fsom_08 0.55 0.23 5.09 1.18 -0.15 2.81 3.97 4.82 3.99
Head of Data Frame containing manual gates and unsupervised clusters
H3K14ac phH3_S10 H3K4me3 Total.H3 Caspase3 PAX6 H3K27ac H3K9ac FxCycle
Apoptotic Cells 0.16 0.21 4.05 0.30 4.70 0.90 2.12 2.13 3.52
fsom_02 0.45 0.24 4.80 0.80 0.03 1.25 3.00 4.12 3.57
Other/G0/G1 0.46 0.23 4.90 0.93 -0.03 1.25 3.25 4.26 3.64
fsom_09 0.78 0.54 5.39 1.68 -0.38 3.25 4.67 5.35 4.20
Other/G2 0.71 0.27 5.35 1.24 -0.31 1.35 4.01 5.05 4.36
Other/Mitosis 1.74 5.42 5.55 1.31 -0.38 1.43 4.10 4.78 4.55

Features MFI by fsom clusters

Hierarchical Clustering (hclust)

Initial assessment through hierarchical clustering

Show code
# Define color palette 
col_1 = colorRamp2(c(-4, 0, 4), hcl_palette = "RdYlBu", reverse = TRUE)
col_1(seq(-4, 4))
[1] "#324DA0FF" "#008EA8FF" "#69BBABFF" "#C9DDC9FF" "#FEFDBEFF" "#F4D481FF"
[7] "#EB9D00FF" "#DE5400FF" "#A51122FF"
Show code
# Sort dendogram nodes
row_dend = dendsort(hclust(dist(df_m)))
col_dend = dendsort(hclust(dist(t(df_m))))

# Plot heatmap
h1 <- Heatmap(df_m, name = "MFI",
              row_title_rot = 0,
              row_names_gp = gpar(fontsize = 8),  # Set row name font size
              column_names_gp = gpar(fontsize = 8),  # Set column name font size
              col = col_1, 
              rect_gp = gpar(col = "white", lwd = 0.5),
              cluster_rows = row_dend,
              cluster_columns = col_dend,
              row_split = 2, column_split = 2,
              row_gap = unit(0.5, "mm"), column_gap = unit(0.5, "mm"), border = TRUE, 
              column_title = "MFI Values Across NPC Clusters Identified by FlowSOM | hclust",
              column_title_gp = gpar(fontsize = 8, fontface = "bold"),
              cell_fun = function(j, i, x, y, width, height, fill) {
                grid.text(sprintf("%.1f", df_m[i, j]), x, y, gp = gpar(fontsize = 5)) # Add text inside cells
              })

h1
Figure 3: Clustered heatmap. Hierarchical clustering
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Pearson Correlation

Useful to cluster data based on the correlation between features rather than their absolute differences. It’s particularly good for identifying clusters with similar expression patterns across different markers, regardless of absolute values.

Show code
# Define color palette 
col_1 = colorRamp2(c(-4, 0, 4), hcl_palette = "RdYlBu", reverse = TRUE)
col_1(seq(-4, 4))
[1] "#324DA0FF" "#008EA8FF" "#69BBABFF" "#C9DDC9FF" "#FEFDBEFF" "#F4D481FF"
[7] "#EB9D00FF" "#DE5400FF" "#A51122FF"
Show code
# Plot heatmap
# Create the heatmap with Pearson correlation as the distance metric
set.seed(135)
h2 <- Heatmap(df_m, name = "MFI",
              clustering_distance_rows = "pearson",  # Use Pearson correlation for row clustering
              clustering_method_rows = "complete", # Specify the clustering method (e.g., complete linkage)
              row_km = 4, row_km_repeats = 100,
              row_title_rot = 0,
              row_names_gp = gpar(fontsize = 9),  # Set row name font size
              column_names_gp = gpar(fontsize = 9),  # Set column name font size
              col = col_1, 
              row_gap = unit(1, "mm"), border = TRUE, 
              column_title = "MFI Values Across NPC Clusters Identified by FlowSOM | pearson",
              column_title_gp = gpar(fontsize = 8, fontface = "bold"),
              cell_fun = function(j, i, x, y, width, height, fill) {
                grid.text(sprintf("%.1f", df_m[i, j]), x, y, gp = gpar(fontsize = 7)) # Add text inside cells
              })
h2
Figure 3: Clustered heatmap. Pearson Correlation
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                2 
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Features MFI in manual gates

Show code
# Define color palette 
col_fall = colorRamp2(c(-3, 0, 3), hcl_palette = "Fall")
col_fall(seq(-4, 4))
[1] "#3C5941FF" "#3C5941FF" "#758A68FF" "#B4BD93FF" "#FBF2C4FF" "#E3C28DFF"
[7] "#D48E53FF" "#C7522BFF" "#C7522BFF"
Show code
# Sort dendogram nodes
row_dend = dendsort(hclust(dist(dfg_m)))
col_dend = dendsort(hclust(dist(t(dfg_m))))

# Plot heatmap
h1g <- Heatmap(dfg_m, name = "MFI", 
              col = col_fall, 
              rect_gp = gpar(col = "white", lwd = 0.5),
              cluster_rows = row_dend,
              cluster_columns = col_dend,
              row_split = 7, column_split = 2,
              row_gap = unit(1, "mm"), column_gap = unit(1, "mm"), border = TRUE,
              column_title = "NPC MFI by manual gating | hclust")

h1g
Figure 4: Clustered heatmap. Manual gates
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                2 
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This data viz strategy aims to highlight the contributions of each feature to different filters (aka clusters).

Prepare data

Show code
# Prepare data
cbp <- data_long


# Convert relevant columns to factors
cbp <- cbp %>%
  mutate(
    feature = factor(feature),
    clusterid = factor(clusterid),
    event = factor(event),
    mfi_shifted = mfi + abs(min(mfi)) + 1  # Shift MFI (not scaled) values to avoid negatives
  )

summary_cbp <- summary(cbp)
kable(summary_cbp, format = "html", caption = "Summary of data containing shifted values")
Summary of data containing shifted values
event clusterid feature mfi mfi_shifted
2595 : 27 fsom_06:182997 Caspase3: 61570 Min. :-1.9451 Min. :1.000
2598 : 27 fsom_08:116640 FxCycle : 61570 1st Qu.: 0.4495 1st Qu.:3.395
2600 : 27 fsom_01: 88902 H3K14ac : 61570 Median : 2.2822 Median :5.227
2602 : 27 fsom_02: 83502 H3K27ac : 61570 Mean : 2.3179 Mean :5.263
2604 : 27 fsom_09: 39312 H3K4me3 : 61570 3rd Qu.: 4.1336 3rd Qu.:7.079
2605 : 27 fsom_05: 19179 H3K9ac : 61570 Max. : 6.8623 Max. :9.807
(Other):553968 (Other): 23598 (Other) :184710 NA NA

Features contributions to each cluster

To assess circular barplots for all clusters, please go to the output folder.

Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters
Figure 5: Features’ contribution to fsom clusters

Goal: Assessment of possible correlations between cell cycle progression, DNA content, H3 content and lineage markers

Correlation between phS10-H3 and DNA content

Goal: Assess phS10H3 and DNA content dynamics

Show code
crp <- data # bring back data without re-shaping to long format

# filter features of interest
filtered_data <- crp %>%
  select(FxCycle, phH3_S10, `Total H3`, clusterid, PAX6) %>%
  rename(H3 = `Total H3`)

# Step 1: Calculate mean PAX6 intensity and correlation for each cluster
cluster_stats <- filtered_data %>%
  group_by(clusterid) %>%
  summarize(mean_PAX6 = mean(PAX6, na.rm = TRUE),
            correlation = cor(FxCycle, phH3_S10, method = "pearson")) %>%
  arrange(desc(mean_PAX6), correlation)  # Sort by mean PAX6 (descending) and then by correlation

# Step 2: Reorder the clusterid factor based on combined ordering
filtered_data <- filtered_data %>%
  mutate(clusterid = factor(clusterid, levels = cluster_stats$clusterid))

# Step 3: Merge the correlation data with the filtered data
filtered_data <- filtered_data %>%
  left_join(cluster_stats, by = "clusterid")

# Step 4: Create the plot with the Pearson correlation as a label inside each facet
bub1 <- filtered_data %>%
  arrange(desc(H3)) %>%
  ggplot(aes(x = FxCycle, y = phH3_S10, size = H3, fill = PAX6)) +
    geom_point(alpha = 0.4, shape = 21, color = "black") +  # Black contour with shape 21
    scale_size(range = c(-1, 5), name = "H3 Fluorescence") + 
    scale_fill_viridis_c(option = "D", name = "PAX6 Intensity") +  # Fill by PAX6 intensity
    theme_ipsum() +
    labs(
      title="Correlation between DNA content and Phospho S10 H3",
      subtitle="Clusters are ordered by PAX6 intensity and Pearson correlation value",
       ) +
    theme(legend.position = "bottom",
          panel.background = element_rect(fill = "white", color = "white"),
          panel.border = element_rect(fill = NA, color = "darkgray"),
          plot.background = element_rect(fill = "white", color = "white"),
          panel.grid = element_blank(),
          panel.grid.major = element_blank()
       ) +
    ylab("Phospho H3 S10 Fluorescence") +
    xlab("FxCycle (DNA Content)") +
    facet_wrap(~ clusterid) +  # Use scales="free" to adjust each facet independently
    geom_text(aes(label = sprintf("r = %.2f", correlation)),
              x = -Inf, y = Inf, hjust = -0.1, vjust = 1.1, size = 3, color = "black")

bub1
Figure 6: Correlation plots for exploratory analysis of features correlation
Show code
ggsave("output/PNG/correlation_plot1.png", bub1,width=14, height=12)
ggsave(filename = "output/SVG/correlation_plot1.svg", plot = bub1, width = 14, height = 12)

Correlation between lineage marker and Caspase-3

Background: Caspase activity mediates the differentiation of embryonic stem cells

Show code
crp2 <- data # bring back data without re-shaping to long format

# filter features of interest
filtered_data2 <- crp2 %>%
  select(FxCycle, phH3_S10, Caspase3, clusterid, PAX6)

# Step 1: Calculate mean PAX6 intensity and correlation for each cluster
cluster_stats2 <- filtered_data2 %>%
  group_by(clusterid) %>%
  summarize(mean_FxCycle = mean(FxCycle, na.rm = TRUE),
            correlation = cor(Caspase3, PAX6, method = "pearson")) %>%
  arrange(desc(mean_FxCycle), correlation)  # Sort by mean PAX6 (descending) and then by correlation

# Step 2: Reorder the clusterid factor based on combined ordering
filtered_data2 <- filtered_data2 %>%
  mutate(clusterid = factor(clusterid, levels = cluster_stats2$clusterid))

# Step 3: Merge the correlation data with the filtered data
filtered_data2 <- filtered_data2 %>%
  left_join(cluster_stats2, by = "clusterid")

# Step 4: Create the plot with the Pearson correlation as a label inside each facet
bub2 <- filtered_data2 %>%
  arrange(desc(phH3_S10)) %>%
  ggplot(aes(x = PAX6, y = Caspase3, size = FxCycle, fill = phH3_S10)) +
    geom_point(alpha = 0.4, shape = 21, color = "black") +  # Black contour with shape 21
    scale_size(range = c(-1, 5), name = "Cell Cycle") + 
    scale_fill_viridis_c(option = "D", name = "Mitosis | phH3_S10") +  
    theme_ipsum() +
    labs(
      title="Correlation between lineage commitment marker and cleaved-Caspase3",
      subtitle="Clusters are ordered by FxCycle intensity and Person correlation value",
       ) +
    theme(legend.position = "bottom",
          panel.background = element_rect(fill = "white", color = "white"),
          panel.border = element_rect(fill = NA, color = "darkgray"),
          plot.background = element_rect(fill = "white", color = "white"),
          panel.grid = element_blank(),
          panel.grid.major = element_blank()
       ) +
    ylab("Cleaved-Caspase3 Fluorescence") +
    xlab("PAX6 Fluorescense") +
    facet_wrap(~ clusterid) +  # Use scales="free" to adjust each facet independently
    geom_text(aes(label = sprintf("r = %.2f", correlation)),
              x = -Inf, y = Inf, hjust = -0.1, vjust = 1.1, size = 3, color = "black")

bub2
Figure 7: Correlation plots for exploratory analysis of features correlation
Show code
ggsave("output/PNG/correlation_plot2.png", bub2,width=14, height=12)
ggsave(filename = "output/SVG/correlation_plot2.svg", plot = bub2, width = 14, height = 12)

Goal: Assessment of the distributions of each H3-PTM within clusters.

From R Gallery

Density plots

Density plots for fsom clusters

Show code
# Represent all density plots in the same axis
d1 <- df %>%
  filter(feature %in% c("H3K9ac", "H3K14ac", "H3K27ac", "H3K4me3")) %>% 
  ggplot( aes(x=mfi_z, fill=feature)) +
    geom_density( color="#e9ecef", alpha=0.6) +
    scale_fill_manual(values=pal2) +
   facet_wrap(~clusterid, ncol = 3, scales = "free_y") +
  labs(title = "Density Plots: Feature distribution per cluster", x = "Scaled Fluorescence", y = "Density") +
  theme_minimal() +
  theme(legend.position = "bottom",
    plot.title = element_text(size = 12, hjust = 0),
    axis.text.y = element_text(size = 8),
    axis.ticks = element_blank(),
    axis.text = element_text(size = 8),
    legend.title = element_text(size = 10),
    legend.text = element_text(size = 8),
    legend.key.size = unit(0.5, "lines"),  # Reduce size of boxes in legend
    panel.spacing = unit(0.05, "lines"),
    panel.grid = element_blank(),
    strip.text = element_text(size = 8)) +
  labs(fill = "Features")
ggsave("output/PNG/density_plot_1.png", width = 30, height = 15, units = "cm", dpi = 600)
ggsave("output/SVG/density_plot_1.svg", plot = d1, width = 7, height = 7)

Density plots for manual gates

# A tibble: 6 × 4
  event clusterid     feature   mfi
  <dbl> <chr>         <chr>   <dbl>
1  2595 fsom_elbow_09 FxCycle  4.46
2  2596 fsom_elbow_06 FxCycle  4.40
3  2598 fsom_elbow_08 FxCycle  3.97
4  2600 fsom_elbow_08 FxCycle  4.53
5  2601 fsom_elbow_08 FxCycle  4.30
6  2602 fsom_elbow_01 FxCycle  4.28
Show code
# Represent all density plots in the same axis
d1g <- dfg %>%
  filter(feature %in% c("H3K9ac", "H3K14ac", "H3K27ac", "H3K4me3")) %>% 
  ggplot( aes(x=mfi_z, fill=feature)) +
    geom_density( color="#e9ecef", alpha=0.6) +
    scale_fill_manual(values=pal2) +
   facet_wrap(~clusterid, ncol = 3, scales = "free_y") +
  labs(title = "Density Plots: Feature distribution per cluster", x = "Scaled Fluorescence", y = "Density") +
  theme_minimal() +
  theme(legend.position = "bottom",
    plot.title = element_text(size = 12, hjust = 0),
    axis.text.y = element_text(size = 8),
    axis.ticks = element_blank(),
    axis.text = element_text(size = 8),
    legend.title = element_text(size = 10),
    legend.text = element_text(size = 8),
    legend.key.size = unit(0.5, "lines"),  # Reduce size of boxes in legend
    panel.spacing = unit(0.05, "lines"),
    panel.grid = element_blank(),
    strip.text = element_text(size = 8)) +
  labs(fill = "Features")
ggsave("output/PNG/density_gate_plot_1.png", width = 30, height = 15, units = "cm", dpi = 600)
ggsave("output/SVG/density_gate_plot_1.svg", plot = d1, width = 7, height = 7)

Histograms

From R Gallery

Histograms allow direct visualization of cell count.

Histograms for fsom clusters

Show code
histogram1 <- df %>%
  filter(feature %in% c("H3K9ac", "H3K14ac", "H3K27ac", "H3K4me3")) %>%
  ggplot(aes(x = mfi, fill = feature)) +
  geom_histogram(binwidth = 0.2, color = "#e9ecef", alpha = 0.6, position = 'identity') +
  scale_fill_manual(values = c("#EDC949", "#B07AA1", "#76B7B2", "#59A14F")) + 
  facet_wrap(~clusterid, ncol = 3, scales = "free_y") +
  labs(title = "Histograms: Feature distribution per cluster", x = "Fluorescence", y = "Cell count") +
  theme_minimal() +
  theme(legend.position = "bottom",
    plot.title = element_text(size = 12, hjust = 0),
    axis.text.y = element_text(size = 6),
    axis.ticks = element_blank(),
    axis.text = element_text(size = 7),
    legend.title = element_text(size = 10),
    legend.text = element_text(size = 8),
    legend.key.size = unit(0.5, "lines"),  # Reduce size of boxes in legend
    panel.spacing = unit(0.05, "lines"),
    panel.grid = element_blank(),
    strip.text = element_text(size = 8)) +
  labs(fill = "Features")
ggsave("output/PNG/histogram_plot_1.png", width = 30, height = 15, units = "cm", dpi = 600)
ggsave("output/SVG/histogram_plot_1.svg", plot = histogram1, width = 7, height = 7)

Histograms for manual gates and fsom clusters

Show code
histogram_gates <- dfg %>%
  filter(feature %in% c("H3K9ac", "H3K14ac", "H3K27ac", "H3K4me3")) %>%
  ggplot(aes(x = mfi, fill = feature)) +
  geom_histogram(binwidth = 0.2, color = "#e9ecef", alpha = 0.6, position = 'identity') +
  scale_fill_manual(values = c("#EDC949", "#B07AA1", "#76B7B2", "#59A14F")) +
  facet_wrap(~clusterid, ncol = 3, scales = "free_y") +
  labs(title = "Histograms: Feature distribution per manual cluster", x = "Fluorescence", y = "Cell count") +
  theme_minimal() +
  theme(legend.position = "bottom",
    plot.title = element_text(size = 12, hjust = 0),
    axis.text.y = element_text(size = 6),
    axis.ticks = element_blank(),
    axis.text = element_text(size = 7),
    legend.title = element_text(size = 10),
    legend.text = element_text(size = 8),
    legend.key.size = unit(0.5, "lines"),  # Reduce size of boxes in legend
    panel.spacing = unit(0.05, "lines"),
    panel.grid = element_blank(),
    strip.text = element_text(size = 8)) +
  labs(fill = "Features")
ggsave("output/PNG/histogram_gates_plot_1.png", width = 30, height = 15, units = "cm", dpi = 600)
ggsave("output/SVG/histogram_gates_plot_1.svg", plot = histogram_gates, width = 7, height = 7)

Violin plots

Goal: To assess the distribution of features, median fluorescence intensity values, and effect sizes within each fsom cluster

To make this plots I used ggstatsplot. I have to admit this package has my favorite vignette of all times.

Pairwise comparisons

Extracting pairwise comparisons (post-hoc test = Dunn)

Pairwise Comparisons of Selected Features per Cluster
group1 group2 statistic p.value alternative distribution p.adjust.method test expression ClusterID Significance
FxCycle H3K14ac 74.7294860 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
FxCycle H3K27ac 7.7758838 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
FxCycle H3K4me3 87.7589100 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
FxCycle H3K9ac 50.0082906 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
FxCycle phH3_S10 103.7220277 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K14ac H3K27ac 66.9536022 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K14ac H3K4me3 162.4883960 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K14ac H3K9ac 124.7377766 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K14ac phH3_S10 28.9925416 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K27ac H3K4me3 95.5347938 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K27ac H3K9ac 57.7841744 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K27ac phH3_S10 95.9461439 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K4me3 H3K9ac 37.7506194 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K4me3 phH3_S10 191.4809376 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
H3K9ac phH3_S10 153.7303182 0.0000000 two.sided z FDR Dunn list, it.... fsom_01 Less than 0.05
FxCycle H3K14ac 84.6724380 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
FxCycle H3K27ac 38.2722283 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
FxCycle H3K4me3 76.1467092 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
FxCycle H3K9ac 36.0680640 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
FxCycle phH3_S10 114.2927835 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K14ac H3K27ac 46.4002096 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K14ac H3K4me3 160.8191472 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K14ac H3K9ac 120.7405019 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K14ac phH3_S10 29.6203455 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K27ac H3K4me3 114.4189375 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K27ac H3K9ac 74.3402923 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K27ac phH3_S10 76.0205551 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K4me3 H3K9ac 40.0786452 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K4me3 phH3_S10 190.4394926 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
H3K9ac phH3_S10 150.3608474 0.0000000 two.sided z FDR Dunn list, it.... fsom_02 Less than 0.05
FxCycle H3K14ac 45.5753898 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
FxCycle H3K27ac 15.9197734 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
FxCycle H3K4me3 4.1626717 0.0000363 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
FxCycle H3K9ac 17.1226307 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
FxCycle phH3_S10 42.1569319 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K14ac H3K27ac 29.6556164 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K14ac H3K4me3 49.7380616 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K14ac H3K9ac 28.4527592 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K14ac phH3_S10 3.4184579 0.0006748 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K27ac H3K4me3 20.0824451 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K27ac H3K9ac 1.2028573 0.2290316 two.sided z FDR Dunn list, it.... fsom_03 More than 0.05
H3K27ac phH3_S10 26.2371585 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K4me3 H3K9ac 21.2853024 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K4me3 phH3_S10 46.3196036 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
H3K9ac phH3_S10 25.0343012 0.0000000 two.sided z FDR Dunn list, it.... fsom_03 Less than 0.05
FxCycle H3K14ac 23.7983484 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
FxCycle H3K27ac 6.5661389 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
FxCycle H3K4me3 24.0270817 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
FxCycle H3K9ac 13.2262823 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
FxCycle phH3_S10 31.5408809 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K14ac H3K27ac 17.2322095 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K14ac H3K4me3 47.8254300 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K14ac H3K9ac 37.0246307 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K14ac phH3_S10 7.7425325 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K27ac H3K4me3 30.5932206 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K27ac H3K9ac 19.7924212 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K27ac phH3_S10 24.9747420 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K4me3 H3K9ac 10.8007993 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K4me3 phH3_S10 55.5679625 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
H3K9ac phH3_S10 44.7671632 0.0000000 two.sided z FDR Dunn list, it.... fsom_04 Less than 0.05
FxCycle H3K14ac 28.1543469 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
FxCycle H3K27ac 6.4800483 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
FxCycle H3K4me3 44.2617008 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
FxCycle H3K9ac 34.6230910 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
FxCycle phH3_S10 42.2340019 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K14ac H3K27ac 34.6343951 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K14ac H3K4me3 72.4160477 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K14ac H3K9ac 62.7774378 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K14ac phH3_S10 14.0796550 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K27ac H3K4me3 37.7816525 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K27ac H3K9ac 28.1430427 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K27ac phH3_S10 48.7140502 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K4me3 H3K9ac 9.6386098 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K4me3 phH3_S10 86.4957027 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
H3K9ac phH3_S10 76.8570929 0.0000000 two.sided z FDR Dunn list, it.... fsom_05 Less than 0.05
FxCycle H3K14ac 118.7494958 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
FxCycle H3K27ac 38.0670250 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
FxCycle H3K4me3 118.7578805 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
FxCycle H3K9ac 65.9219087 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
FxCycle phH3_S10 157.2211576 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K14ac H3K27ac 80.6824708 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K14ac H3K4me3 237.5073763 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K14ac H3K9ac 184.6714044 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K14ac phH3_S10 38.4716619 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K27ac H3K4me3 156.8249055 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K27ac H3K9ac 103.9889336 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K27ac phH3_S10 119.1541326 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K4me3 H3K9ac 52.8359719 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K4me3 phH3_S10 275.9790382 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
H3K9ac phH3_S10 223.1430663 0.0000000 two.sided z FDR Dunn list, it.... fsom_06 Less than 0.05
FxCycle H3K14ac 19.6168753 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
FxCycle H3K27ac 7.4735678 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
FxCycle H3K4me3 10.2436048 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
FxCycle H3K9ac 0.9930648 0.3435840 two.sided z FDR Dunn list, it.... fsom_07 More than 0.05
FxCycle phH3_S10 20.4790651 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K14ac H3K27ac 12.1433075 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K14ac H3K4me3 29.8604801 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K14ac H3K9ac 18.6238105 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K14ac phH3_S10 0.8621898 0.3885831 two.sided z FDR Dunn list, it.... fsom_07 More than 0.05
H3K27ac H3K4me3 17.7171726 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K27ac H3K9ac 6.4805029 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K27ac phH3_S10 13.0054973 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K4me3 H3K9ac 11.2366696 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K4me3 phH3_S10 30.7226699 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
H3K9ac phH3_S10 19.4860003 0.0000000 two.sided z FDR Dunn list, it.... fsom_07 Less than 0.05
FxCycle H3K14ac 82.9395890 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
FxCycle H3K27ac 2.7064980 0.0067997 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
FxCycle H3K4me3 95.9092415 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
FxCycle H3K9ac 71.8563827 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
FxCycle phH3_S10 113.3838465 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K14ac H3K27ac 80.2330910 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K14ac H3K4me3 178.8488305 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K14ac H3K9ac 154.7959717 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K14ac phH3_S10 30.4442575 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K27ac H3K4me3 98.6157394 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K27ac H3K9ac 74.5628807 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K27ac phH3_S10 110.6773485 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K4me3 H3K9ac 24.0528588 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K4me3 phH3_S10 209.2930879 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
H3K9ac phH3_S10 185.2402292 0.0000000 two.sided z FDR Dunn list, it.... fsom_08 Less than 0.05
FxCycle H3K14ac 40.5052181 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
FxCycle H3K27ac 19.5108475 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
FxCycle H3K4me3 62.5408745 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
FxCycle H3K9ac 58.0886943 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
FxCycle phH3_S10 51.3103343 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K14ac H3K27ac 60.0160655 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K14ac H3K4me3 103.0460926 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K14ac H3K9ac 98.5939124 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K14ac phH3_S10 10.8051162 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K27ac H3K4me3 43.0300271 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K27ac H3K9ac 38.5778468 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K27ac phH3_S10 70.8211818 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K4me3 H3K9ac 4.4521802 0.0000085 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K4me3 phH3_S10 113.8512089 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
H3K9ac phH3_S10 109.3990286 0.0000000 two.sided z FDR Dunn list, it.... fsom_09 Less than 0.05
FxCycle H3K14ac 21.0411273 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
FxCycle H3K27ac 6.7941547 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
FxCycle H3K4me3 22.9498481 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
FxCycle H3K9ac 4.1446266 0.0000365 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
FxCycle phH3_S10 20.2749543 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K14ac H3K27ac 14.2469726 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K14ac H3K4me3 43.9909754 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K14ac H3K9ac 25.1857539 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K14ac phH3_S10 41.3160816 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K27ac H3K4me3 29.7440028 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K27ac H3K9ac 10.9387813 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K27ac phH3_S10 27.0691090 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K4me3 H3K9ac 18.8052215 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K4me3 phH3_S10 2.6748938 0.0074753 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05
H3K9ac phH3_S10 16.1303277 0.0000000 two.sided z FDR Dunn list, it.... fsom_10 Less than 0.05

Gating Strategies

Goal: Compare gating strategies to assess whether H3 biogenesis through cell cycle is driving differences in H3-PTMs

Data with manual gates * Gating strategy 1: NPC -> cell Cycle phase -> H3-PTM * Gating strategy 2: NPC -> cell Cycle phase -> Total H3 -> H3-PTM

Note

When exporting from OMIQ, it’s important to export data filter by filter so that no events are dropped.

Head of Data Frame
clusterid feature mfi
Length:700533 Length:700533 Min. :-1.9451
Class :character Class :character 1st Qu.: 0.4653
Mode :character Mode :character Median : 2.6070
NA NA Mean : 2.3985
NA NA 3rd Qu.: 4.2062
NA NA Max. : 6.8623
Assess number of events in each gate
Var1 Freq
NPC/G0_G1 273789
NPC/G0_G1/H3 255591
NPC/G2 86922
NPC/G2/H3 75960
NPC/M 4446
NPC/M/H3 3825
Note

Plots should be generated comparing cluster strategies on x, mfi values on y and one plot per feature.

Show code
generate_cluster_plots <- function(H3, output_dir) {
  # Ensure the output directory exists
  if (!dir.exists(output_dir)) {
    dir.create(output_dir, recursive = TRUE)
  }
  
  # Use actual feature names from the data
  feature_id <- unique(H3$feature)
  
  # Create a list to hold the plots
  plot_list_2 <- list()
  
  # Loop over each feature and generate the plot
  for (feature in feature_id) {
    plot_2 <- H3 %>%
      filter(feature == !!feature) %>%
      ggbetweenstats(
        x = clusterid,
        y = mfi,
        type = "np",  # Non-parametric test
        pairwise.display = "none", # Display only significant pairwise comparisons
        p.adjust.method = "fdr", 
        p.value.args = list(digits = 4), # Adjust p-values for multiple tests using this method
        results.subtitle = TRUE,
        ggplot.component = list(
      scale_color_manual(values = c("#F28E2B", "#FF9DA7","#EDC949", "#B07AA1","#76B7B2","#59A14F")))
      ) +
      # Add labels and title
      labs(
        x = "Gating Strategy",
        y = "Fluorescence Intensity",
        title = paste("Gating strategy comparison for:", feature)
      ) + 
      theme( # Customizations
        text = element_text(family = "Times New Roman", size = 8, color = "black"),
        plot.title = element_text(
          family = "Times New Roman", 
          size = 12,
          face = "bold",
          color = "#2a475e"
        ),
        plot.subtitle = element_text(
          family = "Times New Roman", 
          size = 8, 
          face = "bold",
          color = "#1b2838"
        ),
        plot.title.position = "plot",
        axis.text = element_text(size = 8, color = "black"),
        axis.title = element_text(size = 8),
        axis.ticks = element_blank(),
        axis.line = element_line(colour = "grey50"),
        panel.grid = element_line(color = "#b4aea9"),
        panel.grid.minor = element_blank(),
        panel.grid.major.x = element_blank(),
        panel.grid.major.y = element_line(linetype = "dashed"),
        panel.background = element_rect(fill = "white", color = "white"),
        plot.background = element_rect(fill = "white", color = "white")
      )
    
    # Add the plot to a list
    plot_list_2[[feature]] <- plot_2
  
    
    # Save the individual plot as PNG
    ggsave(
      filename = file.path(output_dir, paste0("gate_h3_violin_plot_", feature, ".png")),
      plot = plot_2,
      width = 20, height = 12, units = "cm", dpi = 600
    )
    
    # Save the individual plot as SVG
    ggsave(
      filename = file.path(output_dir, paste0("gate_h3_violin_plot_", feature, ".svg")),
      plot = plot_2,
      width = 20, height = 12, units = "cm"
    )
      print(plot_2)
  }
  
  # Combine all plots into a grid with 5 rows (or adjust as needed)
  combined_plot_2 <- wrap_plots(plot_list_2, nrow = 5)
  
  # Save the combined plot as PNG
  ggsave(
    filename = file.path(output_dir, "combined_h3_gate_violin_plots.png"),
    plot = combined_plot_2,
    width = 40, height = 50, units = "cm", dpi = 600
  )
  
  # Save the combined plot as SVG
  ggsave(
    filename = file.path(output_dir, "combined_h3_gate_violin_plots.svg"),
    plot = combined_plot_2,
    width = 40, height = 50, units = "cm"
  )
}

# Example usage:
output_directory <- "output/gate_violin_plots"
generate_cluster_plots(H3, output_directory)

Pairwise comparison stats

Pairwise Comparisons of gating strategies for H3K27ac
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 6.4838987 0.0000000 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/G2 NPC/G2/H3 5.3254498 0.0000002 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/M NPC/M/H3 0.8112492 0.4814107 two.sided z FDR Dunn list, it.... More than 0.05
Pairwise Comparisons of gating strategies for H3K9ac
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 3.6382897 0.0003167 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/G2 NPC/G2/H3 1.2898755 0.2111720 two.sided z FDR Dunn list, it.... More than 0.05
NPC/M NPC/M/H3 0.3223694 0.7471729 two.sided z FDR Dunn list, it.... More than 0.05
Pairwise Comparisons of gating strategies for H3K14ac
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 1.0476738 0.3401411 two.sided z FDR Dunn list, it.... More than 0.05
NPC/G2 NPC/G2/H3 0.9923511 0.3439567 two.sided z FDR Dunn list, it.... More than 0.05
NPC/M NPC/M/H3 0.0191648 0.9847096 two.sided z FDR Dunn list, it.... More than 0.05
Pairwise Comparisons of gating strategies for H3K4me3
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 5.401929 0.0000001 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/G2 NPC/G2/H3 3.440348 0.0006225 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/M NPC/M/H3 0.123544 0.9016764 two.sided z FDR Dunn list, it.... More than 0.05
Pairwise Comparisons of gating strategies for PAX6
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 1.4732835 0.1507228 two.sided z FDR Dunn list, it.... More than 0.05
NPC/G2 NPC/G2/H3 2.0135557 0.0508341 two.sided z FDR Dunn list, it.... More than 0.05
NPC/M NPC/M/H3 0.0354808 0.9716964 two.sided z FDR Dunn list, it.... More than 0.05
Pairwise Comparisons of gating strategies for Total H3
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 7.262977 0.0000000 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/G2 NPC/G2/H3 7.969032 0.0000000 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/M NPC/M/H3 2.347042 0.0236539 two.sided z FDR Dunn list, it.... Less than 0.05
Pairwise Comparisons of gating strategies for phH3_S10
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 0.8198298 0.4417641 two.sided z FDR Dunn list, it.... More than 0.05
NPC/G2 NPC/G2/H3 3.6660592 0.0002842 two.sided z FDR Dunn list, it.... Less than 0.05
NPC/M NPC/M/H3 0.0198941 0.9841279 two.sided z FDR Dunn list, it.... More than 0.05
Pairwise Comparisons of gating strategies for FxCycle
group1 group2 statistic p.value alternative distribution p.adjust.method test expression p_comparison
NPC/G0_G1 NPC/G0_G1/H3 0.6556312 0.5908401 two.sided z FDR Dunn list, it.... More than 0.05
NPC/G2 NPC/G2/H3 0.1031999 0.9833618 two.sided z FDR Dunn list, it.... More than 0.05
NPC/M NPC/M/H3 0.0160008 0.9872337 two.sided z FDR Dunn list, it.... More than 0.05

Drug treatments

Drug treatment assays to assess sensitivity of EpiFlow identification in both, spectral and conventional flow cytometry

Summary of spectral flow cytometry experiment

Summary of experimental design
Cell line WTC11
Differentiation Neuronal progenitor cells (NPC)
Treatment 1 DMSO control
Treatment 2

Trichostatin A (TSA) (0.5, 1 and 10 µM)

HDAC inhibitor, showed to increase H3K27ac (PMID: 23093607). Expected outcome: increase in acetylation marks
Treatment 3

A-485 (1, 10 and 20 µM)

p300/CBP inhibitor, showed to decrease H3K27ac (PMID: 28953875). Expected outcome: decreased acetylation marks.
Replicates 3 biological replicates per experimental group
Head of Data Frame
event H3K9ac H3K14ac H3K4me1 H3K27ac gate treatment
4629 5.0134 0.532710 4.4401 6.4594 Mitosis DMSO
4630 5.6764 -0.039929 4.0351 6.4341 G2 DMSO
4634 5.1459 -0.059104 5.0237 6.8079 G0/G1 DMSO
4635 5.2293 0.361370 5.6113 7.2979 NPC | PAX6+ DMSO
4637 5.0843 0.049410 4.1713 6.5476 Other DMSO
4638 5.3414 0.081570 4.2808 6.6148 G0/G1 DMSO
Head of Data Long Format
event gate treatment feature mfi
4629 Mitosis DMSO H3K9ac 5.0134
4630 G2 DMSO H3K9ac 5.6764
4634 G0/G1 DMSO H3K9ac 5.1459
4635 NPC | PAX6+ DMSO H3K9ac 5.2293
4637 Other DMSO H3K9ac 5.0843
4638 G0/G1 DMSO H3K9ac 5.3414
Summary of Data Frame
event gate treatment feature mfi
Min. : 1112 Length:1551252 Length:1551252 Length:1551252 Min. :-3.924
1st Qu.: 52931 Class :character Class :character Class :character 1st Qu.: 2.358
Median :100295 Mode :character Mode :character Mode :character Median : 4.597
Mean :120741 NA NA NA Mean : 4.063
3rd Qu.:157830 NA NA NA 3rd Qu.: 5.816
Max. :583396 NA NA NA Max. : 7.609
Summary of Data Frame after scaling
event gate treatment feature mfi mfi_z.V1
Min. : 1112 Length:1551252 Length:1551252 Length:1551252 Min. :-3.924 Min. :-3.601487
1st Qu.: 52931 Class :character Class :character Class :character 1st Qu.: 2.358 1st Qu.:-0.768861
Median :100295 Mode :character Mode :character Mode :character Median : 4.597 Median : 0.240759
Mean :120741 NA NA NA Mean : 4.063 Mean : 0.000000
3rd Qu.:157830 NA NA NA 3rd Qu.: 5.816 3rd Qu.: 0.790276
Max. :583396 NA NA NA Max. : 7.609 Max. : 1.598658
Head of dataset
event gate treatment feature mfi mfi_z
4629 Mitosis DMSO H3K9ac 5.0134 0.4283355
4630 G2 DMSO H3K9ac 5.6764 0.7272851
4634 G0/G1 DMSO H3K9ac 5.1459 0.4880803
4635 NPC | PAX6+ DMSO H3K9ac 5.2293 0.5256857
4637 Other DMSO H3K9ac 5.0843 0.4603046
4638 G0/G1 DMSO H3K9ac 5.3414 0.5762321

Assess spectral flow sensitivity on fluorescent intensity values distributions per feature within each cell cycle phase

Assess spectral flow sensitivity on fluorescent intensity values distributions of all features within each cell cycle phase

Statistical analysis to assess the effect of drug treatments in each feature

Pairwise comparisons

Extracting pairwise comparisons

Pairwise Comparisons
group1 group2 statistic p.value alternative distribution p.adjust.method test expression Gate Feature Significance
A485 10um A485 20um 0.9068690 0.3826999 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac More than 0.05
A485 10um TSA 05um 84.8632708 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 10um TSA 10um 97.4909448 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 10um TSA 1um 81.5102892 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 1um A485 10um 16.8927188 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 1um A485 20um 16.5865824 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 1um TSA 05um 70.9631481 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 1um TSA 10um 80.5748676 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 1um TSA 1um 67.5962834 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 20um TSA 05um 85.3722052 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 20um TSA 10um 98.4496342 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 20um TSA 1um 81.9927977 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
DMSO A485 10um 70.4285761 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
DMSO A485 1um 43.1838364 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
DMSO A485 20um 72.6809904 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
DMSO TSA 05um 49.6393166 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
DMSO TSA 10um 57.7421748 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
DMSO TSA 1um 45.8513140 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
TSA 05um TSA 10um 0.2048217 0.8377114 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac More than 0.05
TSA 05um TSA 1um 3.2014716 0.0015112 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
TSA 1um TSA 10um 3.6672221 0.0002861 two.sided z FDR Dunn list, it.... G0/G1 H3K9ac Less than 0.05
A485 10um A485 20um 2.5107497 0.0126499 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 10um TSA 05um 59.7950434 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 10um TSA 10um 63.1191182 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 10um TSA 1um 43.4845316 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 1um A485 10um 8.1836971 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 1um A485 20um 10.6480856 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 1um TSA 05um 62.4830194 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 1um TSA 10um 65.6138278 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 1um TSA 1um 46.8395016 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 20um TSA 05um 59.0986256 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 20um TSA 10um 62.4931032 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 20um TSA 1um 42.5884932 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
DMSO A485 10um 84.2061306 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
DMSO A485 1um 82.3460084 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
DMSO A485 20um 85.3068318 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
DMSO TSA 05um 16.3423420 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
DMSO TSA 10um 13.3078276 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
DMSO TSA 1um 0.9175614 0.3588486 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac More than 0.05
TSA 05um TSA 10um 3.9268720 0.0000951 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
TSA 05um TSA 1um 13.0209436 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
TSA 1um TSA 10um 10.1242249 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K27ac Less than 0.05
A485 10um A485 20um 2.3413153 0.0201767 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 10um TSA 05um 62.7634521 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 10um TSA 10um 78.6647860 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 10um TSA 1um 66.0529693 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 1um A485 10um 17.4779823 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 1um A485 20um 15.9186700 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 1um TSA 05um 49.3857457 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 1um TSA 10um 62.3096228 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 1um TSA 1um 52.4028698 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 20um TSA 05um 62.1982169 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 20um TSA 10um 78.3951737 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 20um TSA 1um 65.5298308 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
DMSO A485 10um 31.0331017 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
DMSO A485 1um 45.9985348 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
DMSO A485 20um 35.0852026 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
DMSO TSA 05um 81.5004685 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
DMSO TSA 10um 101.3832420 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
DMSO TSA 1um 85.1045584 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
TSA 05um TSA 10um 4.9448644 0.0000009 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
TSA 05um TSA 1um 2.0850595 0.0370639 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
TSA 1um TSA 10um 2.7230357 0.0071494 two.sided z FDR Dunn list, it.... G0/G1 H3K14ac Less than 0.05
A485 10um A485 20um 0.1215996 0.9032161 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 More than 0.05
A485 10um TSA 05um 21.7737091 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 10um TSA 10um 32.7407811 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 10um TSA 1um 18.8380361 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 1um A485 10um 7.9166609 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 1um A485 20um 8.0418925 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 1um TSA 05um 25.8202400 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 1um TSA 10um 36.6169540 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 1um TSA 1um 23.0313347 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 20um TSA 05um 22.1039766 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 20um TSA 10um 33.3389369 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 20um TSA 1um 19.1380148 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
DMSO A485 10um 28.0884853 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
DMSO A485 1um 15.9462393 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
DMSO A485 20um 29.2547939 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
DMSO TSA 05um 37.6078279 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
DMSO TSA 10um 51.6579853 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
DMSO TSA 1um 34.7410197 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
TSA 05um TSA 10um 5.6969380 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
TSA 05um TSA 1um 2.4232140 0.0161531 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
TSA 1um TSA 10um 8.3538479 0.0000000 two.sided z FDR Dunn list, it.... G0/G1 H3K4me1 Less than 0.05
A485 10um A485 20um 2.7941016 0.0060718 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 10um TSA 05um 80.8658363 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 10um TSA 10um 76.2856727 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 10um TSA 1um 42.2086798 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 1um A485 10um 1.8455858 0.0717895 two.sided z FDR Dunn list, it.... G2 H3K9ac More than 0.05
A485 1um A485 20um 0.5609244 0.5748490 two.sided z FDR Dunn list, it.... G2 H3K9ac More than 0.05
A485 1um TSA 05um 76.5209725 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 1um TSA 10um 72.6200827 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 1um TSA 1um 41.9331606 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 20um TSA 05um 85.1884288 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 20um TSA 10um 80.2247494 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 20um TSA 1um 43.9638737 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
DMSO A485 10um 15.3429667 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
DMSO A485 1um 15.3199167 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
DMSO A485 20um 19.4421610 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
DMSO TSA 05um 75.8700494 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
DMSO TSA 10um 70.9128563 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
DMSO TSA 1um 36.6019697 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
TSA 05um TSA 10um 1.0901065 0.2894495 two.sided z FDR Dunn list, it.... G2 H3K9ac More than 0.05
TSA 05um TSA 1um 7.5187483 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
TSA 1um TSA 10um 6.6562721 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K9ac Less than 0.05
A485 10um A485 20um 1.1090017 0.2674294 two.sided z FDR Dunn list, it.... G2 H3K27ac More than 0.05
A485 10um TSA 05um 35.5181571 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 10um TSA 10um 28.5173232 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 10um TSA 1um 15.7820135 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 1um A485 10um 9.7870612 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 1um A485 20um 9.1375046 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 1um TSA 05um 41.0208833 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 1um TSA 10um 34.3904411 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 1um TSA 1um 20.5600667 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 20um TSA 05um 37.3243963 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 20um TSA 10um 30.0380480 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 20um TSA 1um 16.4689799 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
DMSO A485 10um 73.6149740 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
DMSO A485 1um 74.5350534 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
DMSO A485 20um 79.0879659 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
DMSO TSA 05um 17.0497707 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
DMSO TSA 10um 22.1933025 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
DMSO TSA 1um 16.5547882 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
TSA 05um TSA 10um 4.7881325 0.0000019 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
TSA 05um TSA 1um 5.8813146 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
TSA 1um TSA 10um 2.4850885 0.0135995 two.sided z FDR Dunn list, it.... G2 H3K27ac Less than 0.05
A485 10um A485 20um 3.2808226 0.0010868 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 10um TSA 05um 68.7503326 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 10um TSA 10um 73.8328384 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 10um TSA 1um 43.7789647 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 1um A485 10um 8.0281691 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 1um A485 20um 5.4002772 0.0000001 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 1um TSA 05um 57.1327854 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 1um TSA 10um 62.4705962 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 1um TSA 1um 38.1585026 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 20um TSA 05um 68.0075270 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 20um TSA 10um 73.1721343 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 20um TSA 1um 42.6490086 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
DMSO A485 10um 51.1876064 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
DMSO A485 1um 53.1796337 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
DMSO A485 20um 57.8902160 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
DMSO TSA 05um 112.8850227 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
DMSO TSA 10um 116.0131946 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
DMSO TSA 1um 67.8580266 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
TSA 05um TSA 10um 6.8265969 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
TSA 05um TSA 1um 0.9918210 0.3212849 two.sided z FDR Dunn list, it.... G2 H3K14ac More than 0.05
TSA 1um TSA 10um 3.7434969 0.0002006 two.sided z FDR Dunn list, it.... G2 H3K14ac Less than 0.05
A485 10um A485 20um 2.7947569 0.0064160 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 10um TSA 05um 4.0013140 0.0000882 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 10um TSA 10um 7.1180601 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 10um TSA 1um 1.9122012 0.0558504 two.sided z FDR Dunn list, it.... G2 H3K4me1 More than 0.05
A485 1um A485 10um 15.9111363 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 1um A485 20um 13.9787850 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 1um TSA 05um 9.4255615 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 1um TSA 10um 19.3078106 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 1um TSA 1um 10.3921201 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 20um TSA 05um 1.9244126 0.0558504 two.sided z FDR Dunn list, it.... G2 H3K4me1 More than 0.05
A485 20um TSA 10um 9.3794993 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 20um TSA 1um 3.2657866 0.0014327 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
DMSO A485 10um 17.6451259 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
DMSO A485 1um 2.3240559 0.0222407 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
DMSO A485 20um 15.4240555 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
DMSO TSA 05um 8.9650477 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
DMSO TSA 10um 20.3039223 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
DMSO TSA 1um 9.8289538 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
TSA 05um TSA 10um 9.4625039 0.0000000 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
TSA 05um TSA 1um 4.1142947 0.0000583 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
TSA 1um TSA 10um 2.4890094 0.0149449 two.sided z FDR Dunn list, it.... G2 H3K4me1 Less than 0.05
A485 10um A485 20um 0.8281081 0.4755442 two.sided z FDR Dunn list, it.... Mitosis H3K9ac More than 0.05
A485 10um TSA 05um 20.7816672 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 10um TSA 10um 20.4472298 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 10um TSA 1um 12.7478251 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 1um A485 10um 2.0665207 0.0479039 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 1um A485 20um 2.7897783 0.0069227 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 1um TSA 05um 17.6993755 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 1um TSA 10um 17.4986960 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 1um TSA 1um 11.3546642 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 20um TSA 05um 21.3701578 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 20um TSA 10um 21.0200700 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
A485 20um TSA 1um 13.1147304 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
DMSO A485 10um 13.6158817 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
DMSO A485 1um 9.7005212 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
DMSO A485 20um 14.4556219 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
DMSO TSA 05um 11.4334613 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
DMSO TSA 10um 11.3496749 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
DMSO TSA 1um 6.9223700 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K9ac Less than 0.05
TSA 05um TSA 10um 0.1978983 0.9133112 two.sided z FDR Dunn list, it.... Mitosis H3K9ac More than 0.05
TSA 05um TSA 1um 0.0267498 0.9786593 two.sided z FDR Dunn list, it.... Mitosis H3K9ac More than 0.05
TSA 1um TSA 10um 0.1638869 0.9133112 two.sided z FDR Dunn list, it.... Mitosis H3K9ac More than 0.05
A485 10um A485 20um 0.0809882 0.9354513 two.sided z FDR Dunn list, it.... Mitosis H3K27ac More than 0.05
A485 10um TSA 05um 17.3792701 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 10um TSA 10um 18.1014039 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 10um TSA 1um 11.0507137 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 1um A485 10um 2.5924872 0.0125061 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 1um A485 20um 2.5192252 0.0145287 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 1um TSA 05um 18.1269990 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 1um TSA 10um 18.8132111 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 1um TSA 1um 12.0129442 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 20um TSA 05um 17.4272753 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 20um TSA 10um 18.1479528 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
A485 20um TSA 1um 11.0842897 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
DMSO A485 10um 14.9100006 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
DMSO A485 1um 15.6062333 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
DMSO A485 20um 14.9749688 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
DMSO TSA 05um 7.0460692 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
DMSO TSA 10um 8.0664961 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
DMSO TSA 1um 4.6521762 0.0000046 two.sided z FDR Dunn list, it.... Mitosis H3K27ac Less than 0.05
TSA 05um TSA 10um 0.9982009 0.3712123 two.sided z FDR Dunn list, it.... Mitosis H3K27ac More than 0.05
TSA 05um TSA 1um 0.3368080 0.7730747 two.sided z FDR Dunn list, it.... Mitosis H3K27ac More than 0.05
TSA 1um TSA 10um 0.3591996 0.7730747 two.sided z FDR Dunn list, it.... Mitosis H3K27ac More than 0.05
A485 10um A485 20um 0.1306105 0.8960834 two.sided z FDR Dunn list, it.... Mitosis H3K14ac More than 0.05
A485 10um TSA 05um 16.0305338 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 10um TSA 10um 16.5993265 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 10um TSA 1um 10.0133440 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 1um A485 10um 1.4495559 0.1931769 two.sided z FDR Dunn list, it.... Mitosis H3K14ac More than 0.05
A485 1um A485 20um 1.3338764 0.2251254 two.sided z FDR Dunn list, it.... Mitosis H3K14ac More than 0.05
A485 1um TSA 05um 13.7641443 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 1um TSA 10um 14.3770130 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 1um TSA 1um 9.0049936 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 20um TSA 05um 15.9257028 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 20um TSA 10um 16.4973125 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
A485 20um TSA 1um 9.9526456 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
DMSO A485 10um 9.2442460 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
DMSO A485 1um 9.5139485 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
DMSO A485 20um 9.3677237 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
DMSO TSA 05um 22.9181571 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
DMSO TSA 10um 23.2924251 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
DMSO TSA 1um 14.0929571 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K14ac Less than 0.05
TSA 05um TSA 10um 0.8398602 0.4678179 two.sided z FDR Dunn list, it.... Mitosis H3K14ac More than 0.05
TSA 05um TSA 1um 0.1450990 0.8960834 two.sided z FDR Dunn list, it.... Mitosis H3K14ac More than 0.05
TSA 1um TSA 10um 0.4392470 0.7300071 two.sided z FDR Dunn list, it.... Mitosis H3K14ac More than 0.05
A485 10um A485 20um 0.9845308 0.3590498 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 10um TSA 05um 0.4190263 0.7089567 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 10um TSA 10um 3.2030658 0.0031727 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
A485 10um TSA 1um 2.1622795 0.0494253 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
A485 1um A485 10um 2.0794499 0.0563640 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 1um A485 20um 1.2154713 0.2769361 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 1um TSA 05um 1.9893842 0.0653223 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 1um TSA 10um 4.5466120 0.0000164 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
A485 1um TSA 1um 3.1287494 0.0036866 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
A485 20um TSA 05um 1.1337897 0.2996966 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 20um TSA 10um 3.8965300 0.0002561 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
A485 20um TSA 1um 2.6015480 0.0177171 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
DMSO A485 10um 11.4426125 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
DMSO A485 1um 7.8003634 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
DMSO A485 20um 10.4093383 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
DMSO TSA 05um 8.5960468 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
DMSO TSA 10um 11.1939693 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
DMSO TSA 1um 7.1438052 0.0000000 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
TSA 05um TSA 10um 2.3236686 0.0352507 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 Less than 0.05
TSA 05um TSA 1um 1.7542374 0.1041992 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
TSA 1um TSA 10um 0.1252149 0.9003534 two.sided z FDR Dunn list, it.... Mitosis H3K4me1 More than 0.05
A485 10um A485 20um 2.8147764 0.0048811 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 10um TSA 05um 248.0654870 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 10um TSA 10um 256.4649292 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 10um TSA 1um 190.1380387 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 1um A485 10um 9.1750964 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 1um A485 20um 11.8753846 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 1um TSA 05um 248.5889284 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 1um TSA 10um 257.3213267 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 1um TSA 1um 188.3156788 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 20um TSA 05um 244.9928702 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 20um TSA 10um 253.2004264 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 20um TSA 1um 188.4114706 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
DMSO A485 10um 27.6360927 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
DMSO A485 1um 22.0432629 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
DMSO A485 20um 29.2732910 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
DMSO TSA 05um 132.0881705 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
DMSO TSA 10um 137.6807469 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
DMSO TSA 1um 93.6639148 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
TSA 05um TSA 10um 9.4243870 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
TSA 05um TSA 1um 65.4839154 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
TSA 1um TSA 10um 74.9189919 0.0000000 two.sided z FDR Dunn list, it.... Other H3K9ac Less than 0.05
A485 10um A485 20um 4.6454246 0.0000034 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 10um TSA 05um 161.2479719 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 10um TSA 10um 170.5619304 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 10um TSA 1um 121.2202114 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 1um A485 10um 26.1926639 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 1um A485 20um 20.7789244 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 1um TSA 05um 196.0399199 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 1um TSA 10um 205.6761861 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 1um TSA 1um 154.8119501 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 20um TSA 05um 162.2434280 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 20um TSA 10um 171.3345497 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 20um TSA 1um 123.1823204 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
DMSO A485 10um 82.4780185 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
DMSO A485 1um 101.0134757 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
DMSO A485 20um 84.8150179 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
DMSO TSA 05um 19.5322093 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
DMSO TSA 10um 25.7038135 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
DMSO TSA 1um 7.2829333 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
TSA 05um TSA 10um 10.2720378 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
TSA 05um TSA 1um 45.1245756 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
TSA 1um TSA 10um 55.4537142 0.0000000 two.sided z FDR Dunn list, it.... Other H3K27ac Less than 0.05
A485 10um A485 20um 5.4404104 0.0000001 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 10um TSA 05um 244.2574564 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 10um TSA 10um 292.5023610 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 10um TSA 1um 261.8417879 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 1um A485 10um 28.2700552 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 1um A485 20um 21.9855433 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 1um TSA 05um 224.1641975 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 1um TSA 10um 274.4327580 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 1um TSA 1um 242.3742550 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 20um TSA 05um 232.5039214 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 20um TSA 10um 279.5975083 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 20um TSA 1um 249.4547336 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
DMSO A485 10um 21.3916080 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
DMSO A485 1um 40.3204100 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
DMSO A485 20um 24.8730148 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
DMSO TSA 05um 180.0306060 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
DMSO TSA 10um 211.3926484 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
DMSO TSA 1um 190.3955041 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
TSA 05um TSA 10um 52.2810563 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
TSA 05um TSA 1um 15.9581817 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
TSA 1um TSA 10um 37.0387938 0.0000000 two.sided z FDR Dunn list, it.... Other H3K14ac Less than 0.05
A485 10um A485 20um 2.7389044 0.0068133 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 10um TSA 05um 2.6635541 0.0081186 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 10um TSA 10um 20.1956815 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 10um TSA 1um 34.7942936 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 1um A485 10um 30.0320374 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 1um A485 20um 26.5022777 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 1um TSA 05um 29.4007036 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 1um TSA 10um 53.1605103 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 1um TSA 1um 3.7085167 0.0002736 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 20um TSA 05um 0.3125997 0.7545848 two.sided z FDR Dunn list, it.... Other H3K4me1 More than 0.05
A485 20um TSA 10um 22.6103581 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
A485 20um TSA 1um 30.9824122 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
DMSO A485 10um 4.8444795 0.0000018 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
DMSO A485 1um 14.8251483 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
DMSO A485 20um 2.9372381 0.0038634 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
DMSO TSA 05um 3.2573467 0.0013892 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
DMSO TSA 10um 18.0555813 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
DMSO TSA 1um 17.3598197 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
TSA 05um TSA 10um 24.5860062 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
TSA 05um TSA 1um 34.6015773 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
TSA 1um TSA 10um 59.4513501 0.0000000 two.sided z FDR Dunn list, it.... Other H3K4me1 Less than 0.05
Show code 
R version 4.4.1 (2024-06-14)
Platform: aarch64-apple-darwin20
Running under: macOS Ventura 13.2.1

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/New_York
tzcode source: internal

attached base packages:
[1] grid      stats     graphics  grDevices datasets  utils     methods  
[8] base     

other attached packages:
 [1] colorRamp2_0.1.0       statsExpressions_1.5.5 kableExtra_1.4.0      
 [4] patchwork_1.2.0        ggstatsplot_0.12.4     ggpubr_0.6.0          
 [7] stringr_1.5.1          xml2_1.3.6             downlit_0.4.4         
[10] svglite_2.1.3          Cairo_1.6-2            circlize_0.4.16       
[13] ggridges_0.5.6         wesanderson_0.3.7      dendextend_1.17.1     
[16] ComplexHeatmap_2.20.0  heatmaply_1.5.0        plotly_4.10.4         
[19] hrbrthemes_0.8.7       seriation_1.5.5        dendsort_0.3.4        
[22] readr_2.1.5            tidyr_1.3.1            ggExtra_0.10.1        
[25] lubridate_1.9.3        viridis_0.6.5          viridisLite_0.4.2     
[28] dplyr_1.1.4            ggplot2_3.5.1         

loaded via a namespace (and not attached):
  [1] RColorBrewer_1.1-3      rstudioapi_0.16.0       jsonlite_1.8.8         
  [4] shape_1.4.6.1           datawizard_0.12.2       correlation_0.8.5      
  [7] magrittr_2.0.3          SuppDists_1.1-9.7       farver_2.1.2           
 [10] rmarkdown_2.27          GlobalOptions_0.1.2     vctrs_0.6.5            
 [13] memoise_2.0.1           paletteer_1.6.0         effectsize_0.8.9       
 [16] rstatix_0.7.2           webshot_0.5.5           htmltools_0.5.8.1      
 [19] curl_5.2.1              broom_1.0.6             BWStest_0.2.3          
 [22] htmlwidgets_1.6.4       cachem_1.1.0            mime_0.12              
 [25] lifecycle_1.0.4         iterators_1.0.14        pkgconfig_2.0.3        
 [28] R6_2.5.1                fastmap_1.2.0           PMCMRplus_1.9.10       
 [31] shiny_1.9.0             clue_0.3-65             digest_0.6.36          
 [34] colorspace_2.1-1        rematch2_2.1.2          S4Vectors_0.42.1       
 [37] crosstalk_1.2.1         labeling_0.4.3          fansi_1.0.6            
 [40] timechange_0.3.0        httr_1.4.7              abind_1.4-5            
 [43] compiler_4.4.1          bit64_4.0.5             fontquiver_0.2.1       
 [46] withr_3.0.0             doParallel_1.0.17       backports_1.5.0        
 [49] carData_3.0-5           highr_0.11              Rttf2pt1_1.3.12        
 [52] ggsignif_0.6.4          MASS_7.3-61             rjson_0.2.21           
 [55] gfonts_0.2.0            tools_4.4.1             httpuv_1.6.15          
 [58] extrafontdb_1.0         glue_1.7.0              promises_1.3.0         
 [61] cluster_2.1.6           generics_0.1.3          gtable_0.3.5           
 [64] tzdb_0.4.0              ca_0.71.1               data.table_1.15.4      
 [67] hms_1.1.3               car_3.1-2               utf8_1.2.4             
 [70] BiocGenerics_0.50.0     ggrepel_0.9.5           foreach_1.5.2          
 [73] pillar_1.9.0            vroom_1.6.5             later_1.3.2            
 [76] gmp_0.7-4               bit_4.0.5               renv_1.0.7             
 [79] tidyselect_1.2.1        registry_0.5-1          fontLiberation_0.1.0   
 [82] miniUI_0.1.1.1          knitr_1.48              fontBitstreamVera_0.1.1
 [85] gridExtra_2.3           IRanges_2.38.1          crul_1.5.0             
 [88] stats4_4.4.1            xfun_0.46               matrixStats_1.3.0      
 [91] stringi_1.8.4           boot_1.3-30             lazyeval_0.2.2         
 [94] yaml_2.3.10             kSamples_1.2-10         evaluate_0.24.0        
 [97] codetools_0.2-20        httpcode_0.3.0          extrafont_0.19         
[100] gdtools_0.3.7           tibble_3.2.1            BiocManager_1.30.23    
[103] multcompView_0.1-10     cli_3.6.3               xtable_1.8-4           
[106] parameters_0.22.1       systemfonts_1.1.0       munsell_0.5.1          
[109] Rcpp_1.0.13             zeallot_0.1.0           png_0.1-8              
[112] parallel_4.4.1          assertthat_0.2.1        bayestestR_0.14.0      
[115] Rmpfr_0.9-5             mvtnorm_1.3-1           scales_1.3.0           
[118] insight_0.20.3          purrr_1.0.2             crayon_1.5.3           
[121] GetoptLong_1.0.5        rlang_1.1.4             TSP_1.2-4